Transplanted Donor Cells Rescue Contra-Lateral … · Only 25% of Nuclei are of Donor Origin! R L Notexin ... Mesoangioblasts and skeletal muscle SP cells ... → Multiple time-point

Transplanted Donor Cells Rescue Contra-LateralChemo-ablated Muscle Bed

MGMT-P140K

WT

Notexin + BCNUDONOR CELLS

(i.m.)

Notexin + BCNUSALINE

(i.m.)

R L

or

O6 B

G (i

.p.)

Only 25% of Nuclei are of Donor Origin!

R L

NotexinBCNUCELLS

Notexin BCNU

NOCELLS

14 Days Post-Transplantation(n = 8 /4)

R L R L

Muscle Injected with MGMT Donor Cells

Muscle Injected with Wild-Type Donor Cells

Transplanted Donor Cells Rescue Contra-LateralChemo-ablated Muscle Bed

Images have been removed for the printing purpose.

Right EDL Left EDL

Rescue of Contra-Lateral Chemo-ablated MuscleBed with Negligible Donor Cell Contribution

D14 Post-Transplantation

R L

NotexinBCNU

MGMT(P140K)CELLS

Notexin BCNU

NOCELLS

Imag

es ha

ve be

en re

moved

for th

e prin

ting p

urpos

e.

Sca-1(

-) / C

D45(-)

Sca-1(

+) / C

D45(-)

Sca-1(-)/CD45(-)/CD34(+)• Expressed by Satellite cellprogenitor cells

• Intramuscular transplantationhas been shown to result insuccessful engraftment

Sca-1(+)/CD45(-)/CD34(+)• Expressed by MuscleDerived Stem Cells (MDSCs)

• Intramuscular injection hasbeen shown to result insuccessful engraftment

• Also expressed byMesoangioblasts and skeletalmuscle SP cells

Heterogeneity within CD34+ve Cells from RegeneratingSkeletal Muscle

Images have been removed for the printing purpose.

Sca-1(

-) / C

D45(-)

Sca-1(

+) / C

D45(-)

CD56(-)

/ ALP(+)

CD144(+

) / CD56

(+)

CD144(+

) / CD56

(-)

CD56(-)/ALP(+)/CD34(+)• Pericyte marker

• Intra-arterial injection hasbeen shown to result insuccessful engraftment indown stream muscles

CD144(+)/CD56(+)/CD34(+)• Myo-endothelial marker

• Successful engraftment uponintra-muscular injection

CD144(+)/CD56(-)/CD34(+)• Endothelial cell marker

Heterogeneity within CD34+ve Cells from RegeneratingSkeletal Muscle

Images have been removed for the printing purpose.

Sca-1(

-) / C

D45(-)

Sca-1(

+) / C

D45(-)

CD56(-)

/ ALP(+)

CD144(+

) / CD56

(+)

CD144(+

) / CD56

(-)

Pax7(+

)

CD133(+

)

Pax7(+)/CD34(+)• Satellite cell marker

• Expressed in both quiescentand proliferating Satellite cells

CD133 (+)/CD34(+)• Endothelial progenitor marker butshown to have myogenecity

• Systemic injection resulted insuccessful engraftment intodownstream muscles

Heterogeneity within CD34+ve Cells from RegeneratingSkeletal Muscle

Images have been removed for the printing purpose.

Sca-1(

-) / C

D45(-)

Sca-1(

+) / C

D45(-)

CD56(-)

/ ALP(+)

CD144(+

) / CD56

(+)

CD144(+

) / CD56

(-)

Pax7(+

)

CD133(+

)

CD11b(+)

CXCR4(+)

CD11b(+)/CD34(+)• Monocyte marker

• Shown to aid in phagocytosis

• Promotes muscle stem cellproliferation and muscleregeneration

CXCR4(+)/CD34(+)• Receptor for SDF-1 and expressedin endothelial progenitors

• SDF-1 is secreted from injuredskeletal muscles

• Has been shown to contribute tomyofiber formation

Heterogeneity within CD34+ve Cells from RegeneratingSkeletal Muscle

Images have been removed for the printing purpose.

Peault et al. (2007) Molecular Therapy

3. Adult (Somatic) Stem Cells - Plasticity?

1. Neural Stem Cells for Blood.2. Bone-Marrow Stem Cells for

Liver, Skin and Neurons.3. Blood Vessel Stem Cells for

Fat and Muscles.

Questions & Future Direction:

• What is the Identity and Location of the Responding Stem Cells ?1. Cells recruited from systemic circulation?

WT

NotexinNO

CELLS

Notexin BCNU

MGMT(P140K)CELLS

GFP+: Near ubiquitous expression of EGFPin cardiac and skeletal muscle

From ‘An improved method of parabiosis’ BunsterE. & Meyer R., Anat Rec 1933;57:339-343.

ParabiosisUNSW ACEC Approval 09/27A

Peripheral blood equilibrium isestablished 2-3 weeks post-surgery

Bunster & Meyer (1933) Anatomical Records

Brack (2007) Science

Parabiosis Model

Conboy (2005) Nature

Parabiosis Model

• Is the Pro-Regenerative Signal Systemic ?• Are the Responding Progenitors Recruited from Circulation ?

WT

NotexinBCNU

NOCELLS

Notexin BCNU

MGMT(P140K)CELLS

GFP+:Near-ubiquitousexpression of EGFP

Imag

es ha

ve be

en re

moved

for th

e prin

ting p

urpos

e.

Parabiosis Model

• Is the Pro-Regenerative Signal Systemic ?• Are the Responding Progenitors Recruited from Circulation ?

WT

NotexinBCNU

NOCELLS

Notexin BCNU

MGMT(P140K)CELLS

GFP+:Near-ubiquitousexpression of EGFP

Day -3 Day 0

Male Donor CellsMGMT(tg/tg)

- Notexin into TA & EDL- 3 Days of regeneration

Selectionof Donor Cells-CD34(+ve) cells

- 6 x 104 donor cells

Systemic Stem Cell Activation Signals

• What is the Identity and Location of the Responding Stem Cells ?1. Cells recruited from systemic circulation?2. Local stem cells activated upon receiving signal in systemic circulation?

WT

NotexinNO

CELLS

Notexin BCNU

MGMT(P140K)CELLS

GFP+: Near ubiquitous expression of EGFPin cardiac and skeletal muscle

Future Directions

• Transplantation of various donor sub-populations.→ Identify population(s) responsible for the systemic pro-regenerative signal

• Candidate chemokine/cytokine & receptor screening→ SDF-1, VEGF, TNF-α, IGF-1 etc.→ Quantitative RT-PCR→ FACS analyses

• Implantation of bio-degradable gels releasing recombinant protein(s)→ Alginate Gel, Atelocollagen, Fmoc-L-Phenylalanine-L-Phenylalanine (Fmoc-Phe-Phe)

• Screening of Candidate Cytokines & Chemokines using qRT-PCR- Honours Project, Animesh Singla→ SDF-1, VEGF, TNF-α, IGF-1 etc.→ Multiple time-point analyses (D1 - D2 - D3 - D4 - D5 - D7 - D14)→ Notexin injured vs Chemo-ablated→ alpha-SG null (dystrophic muscle under long-term de/regeneration cycle)

Work in Progress

• Screening of Candidate Cytokines & Chemokines using qRT-PCR- Honours Project, Animesh Singla→ SDF-1, VEGF, TNF-α, IGF-1 etc.→ Multiple time-point analyses (D1 - D2 - D3 - D4 - D5 - D7 - D14)

Work in Progress

Sca-1(

-) / C

D45(-)

Sca-1(

+) / C

D45(-)

CD56(-)

/ ALP(+)

CD144(+

) / CD56

(+)

CD144(+

) / CD56

(-)

Pax7(+

)

CD133(+

)

CD11b(+)

CXCR4(+)

Images have been removed for the printing purpose.

• Screening of Candidate Cytokines & Chemokines using qRT-PCR- Honours Project, Animesh Singla→ SDF-1, VEGF, TNF-α, IGF-1 etc.→ Multiple time-point analyses (D1 - D2 - D3 - D4 - D5 - D7 - D14)

Work in Progress

Mousegastrocnemius5dayspostfreeze‐injuryshowingVEGF(brown)staining.(Wagatsuma,MolCellBiochem2007)

MusclebiopsyfromDMDpaQent(Christovet.al.,MolBiolCell2007)

• Screening of Candidate Cytokines & Chemokines using qRT-PCR- Honours Project, Animesh Singla→ SDF-1, VEGF, TNF-α, IGF-1 etc.→ Multiple time-point analyses (D1 - D2 - D3 - D4 - D5 - D7 - D14)→ Notexin injured vs Chemo-ablated→ alpha-SG null (dystrophic muscle under long-term de/regeneration cycle)

Work in Progress

alpha‐SGnullDiaphragm

8‐weekold 8‐weekold 16‐weekold(Ducloset.al.,JCellBiol1998)